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WO2000039347A1 - Proteine de suppression de tumeur, impliquee dans la transmission des signaux de mort, et methodes diagnostiques, therapeutiques et de criblage basees sur cette proteine - Google Patents

Proteine de suppression de tumeur, impliquee dans la transmission des signaux de mort, et methodes diagnostiques, therapeutiques et de criblage basees sur cette proteine Download PDF

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Publication number
WO2000039347A1
WO2000039347A1 PCT/US1999/031280 US9931280W WO0039347A1 WO 2000039347 A1 WO2000039347 A1 WO 2000039347A1 US 9931280 W US9931280 W US 9931280W WO 0039347 A1 WO0039347 A1 WO 0039347A1
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casp8
gene
inactivation
caspase
cells
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PCT/US1999/031280
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WO2000039347A9 (fr
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Vincent J. Kidd
Jill M. Lahti
Tai Teitz
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St. Jude Children's Research Hospital
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Priority to AU22220/00A priority Critical patent/AU2222000A/en
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Publication of WO2000039347A9 publication Critical patent/WO2000039347A9/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6472—Cysteine endopeptidases (3.4.22)
    • C12N9/6475—Interleukin 1-beta convertase-like enzymes (3.4.22.10; 3.4.22.36; 3.4.22.63)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/154—Methylation markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/172—Haplotypes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
    • G01N2333/95—Proteinases, i.e. endopeptidases (3.4.21-3.4.99)
    • G01N2333/964—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue
    • G01N2333/96425—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals
    • G01N2333/96427—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general
    • G01N2333/9643—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general with EC number
    • G01N2333/96466—Cysteine endopeptidases (3.4.22)

Definitions

  • the present invention relates to identification of tumor suppressor activity of a protein, and to related diagnostic and therapeutic compositions and methods.
  • the discovery of this tumor suppressor activity provides screening targets as well, particularly screening for compounds that overcome gene inactivation that results from genomic methylation of the promoter.
  • cysteine proteases caspase-8 (alternatively named FLICE1/MACH1), caspase 9 (alternatively named Apaf 3/Mch6) and caspase- 10 (alternatively named FLICE2/MACH 2) are related proteins that share homology with the prototypic member of the larger caspase gene family, interleukin-l ⁇ -converting enzyme (ICE)/caspase 1 (Muzio et al, J. Biol. Chem., 1998, 273:2926-2930; Boldin et al, Cell, 1996, 85:803-815; Srinivasula et al. , J. Biol.
  • FLICE1/MACH1 interleukin-l ⁇ -converting enzyme
  • Caspases 8, 9, and 10 are the only known members of this family that contain duplicated death effector domains (DEDs) in a long pro-domain in their amino terminus that precedes the cysteine protease catalytic domain in their carboxyl terminus.
  • the DEDs allow caspase-8 to interact directly with an adaptor molecule, FADD, that also contains a DED.
  • FADD contains a death domain (DD) that allows it to directly associate with a number of cell death receptors (e.g., Fas, DR3).
  • DD death domain
  • Caspase-8 along with the adapter molecule FADD, is part of the death inducing signaling complex (DISC) associated with the Fas receptor (Medema, et al, EMBO J., 1997, 16:2794-2804). It is activated by autoproteolytic cleavage following recruitment to the receptor through its interaction with the DED of FADD, which allows caspase-8 to form aggregates (Scaffidi et al, EMBO J., 1998, 17:1675-1687; Yang et al, Molecular Cell, 1998, 1:319-325; Muzio et al, supra, 1998).
  • DISC death inducing signaling complex
  • caspases 8 and 9 are located upstream of all other caspases and their effector function is responsible for the activation of the "caspase cascade", and subsequent cell death, that occurs following Fas/DR3/TNFR1 activation and release of cytochrome c from mitochondria.
  • Such important apoptotic signaling molecules may be likely targets for disregulation/alteration during tumorigenesis.
  • the present invention relates to the discovery that CASP8 is inactivated in cancers, and plays a role of a tumor suppressor gene.
  • the invention provides a method for detecting inactivation of a CASP8 gene.
  • the method comprises detecting a modification of genomic DNA comprising the CASP8 gene, particularly the promoter, wherein such a modification results in inactivation of a CASP8 gene.
  • This method is particularly useful for obtaining a diagnosis or prognosis of a cancer, particularly a neuroblastoma.
  • the invention further provides a method for diagnosis or prognosis of a cancer, particularly one associated with amplification of a MYC oncogene, comprising detecting inactivation of a CASP8 gene.
  • inactivation of the CASP8 gene is indicative of the presence of a cancer or a poor prognosis for outcome of treatment of the cancer, at least by conventional therapies.
  • detection of inactivation of CASP8 involves detecting methylation of the CASP8 promoter.
  • the invention further provides a nucleic acid comprising at least a part of the genomic gene encoding CASP8.
  • the nucleic acid is a CASP8 genomic DNA;
  • the CASP8 genomic DNA comprises a nucleic acid sequence as depicted in SEQ ID NOS:3-10
  • the CASP8 promoter comprises a nucleic acid sequence as depicted in SEQ ID NOS: 1-2.
  • the invention provides a kit for detecting inactivation of a CASP8 gene comprising a detection assay, e.g. , an immunoassay, PCR-based assay, or hybridization assay, for inactivation of a CASP8 gene.
  • a kit of the invention provides for detection of the methylation of the CASP8 promoter.
  • the invention provides a method of treating a cancer in a subject. The method comprises administering an amount of a vector that expresses a gene encoding functional CASP8 effective to express a functional level of CASP8 into cells of the subject, i.e., gene therapy.
  • the invention provides a vector, such as a defective virus
  • non- viral vector that expresses a gene encoding functional human CASP8 in human target cells.
  • the method further comprises using such gene therapy in combination with other anti-cancer therapies.
  • the invention preferably comprises using such gene therapy in combination with chemotherapy.
  • CASP8 gene is inactivated with a candidate compound and detecting whether the cell undergoes apoptosis.
  • Salient features to the protein e.g., DEDs, caspase subunits, Asp-X cleavage sites (D-S and D-F), the catalytic cysteine residue (QACXG) (SEQ ID NOJ 1), and the substrate specificity motif (RNPAEGTW) (SEQ ID NO: 12) are also shown.
  • FIGS. 2 A and 2B Southern blot analysis of total genomic DNA from three different neuroblastoma cell lines. Genomic DNA was subjected to restriction endonuclease digestion with either EcoRI or Hindlll and then hybridized with the caspase-8 cDNA. This analysis revealed that both enzymes are associated with RFLPs. The arrows to the right of each panel indicate the different polymorphic fragments that hybridized with the cDNA probe.
  • A With EcoRI, an approximately 4.7 kb fragment loses an EcoRI site, generating an 8.8 kb fragment.
  • B With Hindlll, a 4.0 kb fragment loses a Hindlll restriction site, resulting in a new 7.2 kb fragment.
  • the haplotype of the three cell lines for each of the enzymes is indicated below each panel. Size of genomic DNA in kb is indicated to the left of each panel. Figures 3A, 3B, 3C, and 3D. Fluorescent in situ hybridization (FISH) localization of the human BAC clone containing CASP8.
  • FISH Fluorescent in situ hybridization
  • A FISH on metaphase chromosome spreads with the human genomic BAC clone CASP8.43 labeled with digoxigenin-11-UTP which is visualized with fiuorescein-conjugated avidin (green; denoted by the arrows), and the human genomic chromosome 2 centromeric probe labeled with biotin- 14-d ATP which is visualized with Texas red Avidin (red; denoted by the arrowheads) (color photograph). For comparison, the chromosomes were visualized with DAPI counter staining. An interphase nucleus is also shown to demonstrate that the gene is single copy.
  • B Ideogram of G-banded human chromosome 2 indicating the location of CASP8 (2q33-34).
  • C, D Fiber FISH analysis of the deletion in one of the NB7 alleles (C) and in the EBV-transformed B- cell containing a normal CASP8 allele (D).
  • a PI clone containing only CASP10 green was used as a reference for measurements to compare the length of the CASP8 PI hybridization signal (red) from NB7 with a EBV-transformed B-cell (the CASP8 PI does not contain CASP10).
  • the approximate size of the deletion is indicated.
  • Figures 4A, 4B, 4C, 4D, and 4E Analysis of CASP8 structure and expression in human neuroblastoma cell lines. Heterozygous (+/-) deletions determined by FISH are indicated above the appropriate cell line.
  • A Southern blot analysis of the human CASP8 locus. The variant 8.0 and 4.8 kb EcoRI fragments of CASP8 are polymorphisms.
  • B Southern blot analysis of the human CASP10 locus in the same NB cell lines.
  • C Northern blot analysis of caspase-8 mRNA expression in the same NB cell lines.
  • D Western blot analysis of the caspase-8 and caspase-10 proteins in the various neuroblastoma cell lines from SJCRH (NB1-NB21), Memorial Sloan Kettering (SK), and small-cell lung carcinoma cell lines (NCLH). The blot includes tubulin control for the protein loading quantification.
  • E Immunoblot analysis of N-Myc expression in the NB cell lines.
  • Figure 5 RNase protection analysis of the expression of apoptotic genes in human neuroblastoma cell lines.
  • Figures 6A and 6B Methylation status of CASP8, and its effect on caspase-8 expression, in NB cell lines.
  • B Methylation PCR analysis of the NB cell lines. The methylated primers detect only DNA that contain methylated CpG dinucleotides, whereas the unmethylated primers detect only DNA that contains no methylated CpG dinucleotides.
  • FIGS 7A, 7B, and 7C Analysis of neuroblastoma patient material by methylation PCR and FISH.
  • B Semi-quantitative PCR analysis of SJ-910008 and SJ-890191 patient material for caspase-8 (B) and caspase-9 (C) mRNAs. The presence (+) or absence (-) of reverse transcriptase in the respective reactions is shown above each gel.
  • Figures 8A, 8B, 8C, 8D, and 8E Effects of retro virus expression of caspase-8 in three different NB cell lines that are caspase-8 nulls.
  • A Immunoblot demonstrating that caspase-8 (wild type) and caspase-8(DN) are expressed.
  • the caspase- ⁇ (DN) product migrates at a higher molecular weight due to an HA-tag attached to its carboxyl-terminus.
  • B, C Morphology of NB7/Casp8 cells 5 hours after treatment with TNF ⁇ and cycloheximide.
  • Membrane blebbing Nomarski view, B
  • condensed chromatin visualized by DAPI staining, C
  • D Percentage of cells that are positive for cell surface annexin-V staining, another hallmark of apoptotic cells.
  • E Apoptotic induction by Fas mAb in proliferating NB8 and NB10 cells stably expressing caspase-8 at a low level. Induction measured as percentage of cells that are TUNEL-positive.
  • the various cell lines and their treatment regimens are shown at the bottom.
  • the present invention is based, in part, on the surprising discovery that CASP8 is functionally inactivated in greater than 90% of all MYCN amplified neuroblastoma cell lines analyzed.
  • Neuroblastoma is a pediatric solid tumor, and those cases with MYCN amplification have a particularly poor prognosis.
  • Inactivation of CASP8 was observed to occur by homozygous deletion, heterozygous deletion coupled with gene silencing by methylation, and homozygous gene silencing by methylation.
  • the data further show that the C ASP 10 gene, which is closely linked to CASP8, is not affected in a cell line containing the homozygous deletion.
  • the CASP8 promoter region sequences in particular Region 1 (SEQ ID NOJ) and Region 2 (SEQ ID NO:2), which have not been previously characterized, are crucial to the design and execution of the genomic methylation PCR analysis of CASP8 gene inactivation.
  • Methylation PCR can be used to examine even minute amounts of patient material to demonstrate whether the CASP8 gene expresses an mRNA and protein product. This method, and associated kits, are particularly preferred for evaluating CASP8 inactivation.
  • methylation PCR analysis can be performed with the primers of SEQ ID NOS:29-34, as described in the Examples, infra.
  • the promoter Region 1 sequence is located upstream (5') to exon 1, which is the alternatively-spliced 5 ' untranslated region that is less commonly used.
  • the promoter Region 2 sequence is located downstream (3') of exon 1 and upstream of exon 2, which is the more commonly used 5 1 untranslated region.
  • activation of caspase-8 and “inactivation of CASP8 gene” are used interchangeably to refer to a modification of the genomic sequence of CASP8 that results in impairment of transcription or translation of the gene, or of activity of the gene product. For example, genomic methylation of the CASP8 promoter results in inactivation of that allele.
  • deletion of the segment of the chromosome containing CASP8, i.e., deletion of the CASP8 gene results in inactivation of that allele of the gene. Inactivation of one allele may reduce the level of expression of caspase-8 to below that necessary for proper cellular regulation. No caspase-8 expression occurs with inactivation of both alleles by either or both mechanisms (or any of the other mechanisms discussed herein).
  • modification of genomic DNA refers to any mutation of the DNA that impairs gene expression or protein activity.
  • mutations that lead to insertion of a heterologous sequence in the gene, truncation of the gene, and introduction of a nonsense mutation, a frameshift mutation, a splice-site mutation, or a missense mutation can result in inactivation of the gene.
  • point mutations polymorphisms
  • point mutations can impact mRNA stability and translation efficiency, for example by introducing a base that affects secondary structure of the message.
  • Other point mutations for example in the caspase DED or catalytic domains, can lead to JO- expression of an inactive protein. In the latter circumstance, protein expression may be detectable (e.g., by immunoassay), so only analysis of the CASP8 gene permits identification of an inactivating point mutation.
  • Cancers related to the present invention include solid tumors, including carcinomas, and non-solid tumors, including hematologic malignancies.
  • these tumors overexpress or evidence amplification of a MYC family gene. More preferably, caspase-8 is inactivated in these tumors.
  • solid tumors include sarcomas and carcinomas such as, but not limited to: fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma,
  • Hematologic malignancies include leukemias, lymphomas, and multiple myelomas.
  • the following are non- limiting preferred examples of the cancers that can be diagnosed (including determination of a diagnosis or prognosis, or both) or treated in accordance with the present invention: neuroblastoma (particularly juvenile neuroblastoma), small-cell lung carcinoma, non-small-cell lung carcinoma, colorectal carcinoma, and uterine cervical carcinoma.
  • the invention advantageously involves the identification and characterization of the genomic sequence of CASP8 (SEQ ED NOS:3-10). The results show that the CASP8 gene contains at least 11 exons spanning approximately 30 kb on human chromosome band 2q33-34.
  • Chromosome 2 band q33-34 is also involved in tumorigenesis, with loss of heterogeneity (LOH) being reported in a number of tumors.
  • LH heterogeneity
  • CASP8 The specific following genomic sequences of CASP8 are provided: exon 3, flanked by partial sequences of introns 2 and 3 S ⁇ Q ED NO:3 exon 4, flanked by partial sequences of introns 3 and 4 SEQ D NO:4 exon 5, flanked by partial sequences of introns 4 and 5 SEQ ED NO:5 exon 6, flanked by partial sequences of introns 5 and 6 SEQ ID NO:6 exon 7, flanked by partial sequences of introns 6 and 7 SEQ ID
  • aa amino acids
  • bp base pairs
  • cDNA DNA complementary to RNA
  • DD death domain
  • DED death effector domain
  • DISC death-inducing signaling complex
  • FADD Fas associated death domain
  • FISH fluorescent in situ hybridization
  • ICE interleukin-l ⁇ -converting enzyme
  • GFP green fluorescent protein
  • kb(s) green fluorescent protein
  • nt nucleotide
  • oligo oligodeoxyribonucleotide
  • ORF open reading frame
  • RFLP restriction fragment length polymo ⁇ hism
  • SSC 0J5M NaCl/0.015M Na, citrate pH7.6
  • UTR untranslated region(s).
  • the term “about” or “approximately” means within 20%, preferably within 10%, and more preferably within 5% of a given value or range.
  • the term means within an order of magnitude, and preferably a factor of two, of a value.
  • an isolated nucleic acid includes a PCR product, an isolated mRNA, a cDNA, or a restriction fragment.
  • an isolated nucleic acid is preferably excised from the chromosome in which it may be found, and more preferably is no longer joined to non-regulatory, non-coding regions, or to other genes, located upstream or downstream of the gene contained by the isolated nucleic acid molecule when found in the chromosome.
  • the isolated nucleic acid lacks one or more introns.
  • Isolated nucleic acid molecules can be inserted into plasmids, cosmids, artificial chromosomes, and the like.
  • a recombinant nucleic acid is an isolated nucleic acid.
  • An isolated protein may be associated with other proteins or nucleic acids, or both, with which it associates in the cell, or with cellular membranes if it is a membrane-associated protein.
  • An isolated organelle, cell, or tissue is removed from the anatomical site in which it is found in an organism.
  • An isolated material may be, but need not be, purified.
  • purified refers to material that has been isolated under conditions that reduce or eliminate unrelated materials, i.e., contaminants.
  • a purified protein is preferably substantially free of other proteins or nucleic acids with which it is associated in a cell; a purified nucleic acid molecule is preferably substantially free of proteins or other unrelated nucleic acid molecules with which it can be found within a cell.
  • a purified tumor cell is preferably substantially free of other normal cells.
  • substantially free is used operationally, in the context of analytical testing of the material.
  • purified material substantially free of contaminants is at least 50% pure; more preferably, at least 90% pure, and more preferably still at least 99% pure. Purity can be evaluated by chromatography, gel electrophoresis, immunoassay, composition analysis, biological assay, and other methods known in the art.
  • nucleic acid molecule e.g., CASP8, cDNA, gene, etc.
  • normal text e.g., CASP8 or caspase-8 indicates the polypeptide or protein.
  • the present invention contemplates isolation of a nucleic acids encoding a CASP8 of the invention, including a full length, or naturally occurring form of CASP8, and any antigenic fragments thereof from any source.
  • Other nucleic acids encode all or part of a CASP8 exon or intron, or both.
  • the invention provides a complete and partial CASP8 genomic DNAs (SEQ ID NOS:3-10).
  • clones BAC -43 also termed herein CASP-43) and B AC-44 (also termed herein CASP-44), or clones having similar structure, contain the complete CASP8 genomic gene.
  • the invention also provides CASP8 restriction fragments and restriction fragment polymo ⁇ hisms, including, but by no means limited to, EcoRI fragments of 4.7 kb and about 8.0 kb, and H-wdlll fragments of 4.0 kb and about 7.2 kb.
  • Polymo ⁇ hic variants of CASP8, particularly polymo ⁇ hisms (i.e., mutations) that result in inactivation of caspase-8, are also included.
  • the present invention relates to human CASP8 nucleic acids, including allelic variants (e.g., as represented by the RFLPs noted above), in certain embodiments, such as for gene therapy, non-human variants of CASP8 are contemplated.
  • the invention provides Regions 1 (S ⁇ Q ID NOJ) and 2 (S ⁇ Q ID NO:2) of the CASP8 promoter. Considered from the 5' to 3' direction, these two regions flank exon 1 (a 5' UTR that is less commonly used), and exons 1 and 2 (the alternatively spliced 5' UTR that is more commonly used) flank promoter Region 2. Also provided are oligonucleotides that hybridize to the promoters, and particularly to methylated or unmethylated forms of the promoters. In a specific embodiment, these oligonucleotides are PCR primer pairs having sequences as depicted in S ⁇ Q ID NOS:29-34, as described in Example 3, infra.
  • a “vector” is a recombinant nucleic acid construct, such as plasmid, phage genome, virus genome, cosmid, or artificial chromosome, to which another DNA segment may be attached.
  • the vector may bring about the replication of the attached segment, e.g., in the case of a cloning vector.
  • a “replicon” is any genetic element (e.g., plasmid, chromosome, virus) that functions as an autonomous unit of DNA replication in vivo, i.e., it is capable of replication under its own control.
  • the term “vector” includes both viral and nonviral means for introducing the nucleic acid into a cell in vitro, ex vivo or in vivo.
  • Non-viral vectors include plasmids, liposomes, electrically charged lipids (cytofectins), DNA-protein complexes, and biopolymers.
  • Viral vectors include retrovirus, adeno-associated virus, pox, baculovirus, vaccinia, he ⁇ es simplex, Epstein-Barr and adenovirus vectors, as set forth in greater detail below.
  • a vector may also contain one or more regulatory regions, and/or selectable markers useful in selecting, measuring, and monitoring nucleic acid transfer results (transfer to which tissues, duration of expression, etc.).
  • a “cassette” refers to a segment of DNA that can be inserted into a vector at specific restriction sites.
  • the segment of DNA encodes a polypeptide of interest, and the cassette and restriction sites are designed to ensure insertion of the cassette in the proper reading frame for transcription and translation.
  • a cell has been "transfected” by exogenous or heterologous DNA when such DNA has been introduced inside the cell.
  • a cell has been "transformed” by exogenous or heterologous DNA when the transfected DNA is expressed and effects a function or phenotype on the cell in which it is expressed.
  • heterologous refers to a combination of elements not naturally occurring.
  • heterologous DNA refers to DNA not naturally located in the cell, or in a chromosomal site of the cell.
  • a heterologous expression regulatory element is a such an element operatively associated with a different gene than the one it is operatively associated with in nature, such as a CMV promoter operatively associated with a CASP8 coding region.
  • an CASP8 gene is heterologous to vector DNA in which it is inserted for cloning or expression.
  • nucleic acid molecule refers to the phosphate ester polymeric form of ribonucleosides (adenosine, guanosine, uridine or cytidine; "RNA molecules”) or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; "DNA molecules”), or any phosphoester analogs thereof, such as phosphorothioates and thioesters, in either single stranded form, or a double-stranded helix. Double stranded DNA-DNA, DNA- RNA and RNA-RNA helices are possible.
  • nucleic acid molecule refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms.
  • this term includes double-stranded DNA found, inter alia, in linear (e.g., restriction fragments) or circular DNA molecules, plasmids, and chromosomes.
  • sequences may be described herein according to the normal convention of giving only the sequence in the 5' to 3' direction along the nontranscribed strand of DNA (i.e., the strand having a sequence homologous to the mRNA).
  • a “recombinant DNA molecule” is a DNA molecule that has undergone a molecular biological manipulation.
  • the present invention provides antisense nucleic acids (including ribozymes), which may be used as probes or to inhibit expression of CASP8.
  • An "antisense nucleic acid” is a single stranded nucleic acid molecule which, on hybridizing with complementary bases in an RNA or DNA molecule, inhibits the latter's role. If the RNA is a messenger RNA transcript, the antisense nucleic acid is a countertranscript or mRNA-interfering complementary nucleic acid.
  • “antisense” broadly includes RNA-RNA interactions, RNA-DNA interactions, ribozymes, and RNase-H mediated arrest.
  • Antisense nucleic acid molecules can be encoded by a recombinant gene for expression in a cell (e.g., U.S. Patent Nos.
  • 5,814,500 and 5,811,234) can be prepared synthetically (e.g., U.S. Patent No. 5,780,607).
  • oligonucleotides that contain phosphorothioates, phosphotriesters, methyl phosphonates, short chain alkyl, or cycloalkl intersugar linkages or short chain heteroatomic or heterocyclic intersugar linkages.
  • 5,637,684 describes phosphoramidate and phosphorothioamidate oligomeric compounds.
  • oligonucleotides having mo ⁇ holino backbone structures U.S. Pat. No. 5,034,506.
  • the phosphodiester backbone of the oligonucleotide may be replaced with a polyamide backbone, the bases being bound directly or indirectly to the aza nitrogen atoms of the polyamide backbone (Nielsen et al, Science, 1991, 254:1497).
  • oligonucleotides may contain substituted sugar moieties comprising one of the following at the 2' position: OH, SH, SCH 3 , F, OCN, O(CH 2 ) n NH 2 or O(CH 2 ) ⁇ CH 3 where n is from 1 to about 10; C, to C 10 lower alkyl, substituted lower alkyl, alkaryl or aralkyl; Cl; Br; CN; CF 3 ; OCF 3 ; O-; S-, or N-alkyl; O-, S-, or N-alkenyl; SOCH 3 ; SO 2 CH 3 ; ONO 2 ;NO 2 ; N 3 ; NH 2 ; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted sialyl; a fluorescein moiety; an RNA cleaving group; a reporter group; an intercalator; a group for improving the pharmacokinetic properties of an oli
  • Oligonucleotides may also have sugar mimetics such as cyclobutyls or other carbocyclics in place of the pentofuranosyl group.
  • Nucleotide units having nucleosides other than adenosine, cytidine, guanosine, thymidine and uridine may be used, such as inosine.
  • a “gene” is used herein to refer to a portion of a DNA molecule that includes a polypeptide coding sequence operatively associated with expression control sequences.
  • a gene includes both transcribed and untranscribed regions.
  • the transcribed region may include introns, which are spliced out of the mRNA, and 5'- and 3 '-untranslated (UTR) sequences along with protein coding sequences.
  • a gene can be a genomic or partial genomic sequence, in that it contains one or more introns.
  • the term gene may refer to a cDNA molecule (i.e., the coding sequence lacking introns).
  • a DNA "coding sequence” is a double-stranded DNA sequence which is transcribed and translated into a polypeptide in a cell in vitro or in vivo when placed under the control of appropriate regulatory sequences.
  • the boundaries of the coding sequence are determined by a translation start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxyl) terminus.
  • “Expression control sequences” are regulatory sequences that flank a coding sequence, such as promoters, enhancers, suppressors, terminators, and the like, and that provide for the expression of a coding sequence in a host cell.
  • a polyadenylation signal and transcription termination sequence will usually be located 3' to the coding sequence.
  • a ribosome binding site is an expression control sequence.
  • a “promoter sequence” is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence.
  • the promoter sequence is bounded at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background.
  • a transcription initiation site (conveniently defined for example, by mapping with nuclease SI), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase.
  • a coding sequence is "under the control" of transcriptional and translational control sequences in a cell when RNA polymerase transcribes the coding sequence into mRNA, which is then trans-RNA spliced (if it contains introns) and translated into the protein encoded by the coding sequence.
  • a nucleic acid molecule is "hybridizable" to another nucleic acid molecule, such as a cDNA, genomic DNA, or RNA, when a single stranded form of the nucleic acid molecule can anneal to the other nucleic acid molecule under the appropriate conditions of temperature and solution ionic strength (see Sambrook et al, 1989).
  • the conditions of temperature and ionic strength determine the "stringency" of the hybridization.
  • low stringency hybridization conditions corresponding to a T m of 55 °C, can be used, e.g., 5x SSC, 0.1% SDS, 0.25% milk, and no formamide; or 30% formamide, 5x SSC, 0.5% SDS).
  • Moderate stringency hybridization conditions correspond to a higher T m , e.g., 40% formamide, with 5x or 6x SCC.
  • High stringency hybridization conditions correspond to the highest T m , e.g., 50% formamide, 5x or 6x SCC.
  • Hybridization requires that the two nucleic acids contain complementary sequences, although depending on the stringency of the hybridization, mismatches between bases are possible.
  • the appropriate stringency for hybridizing nucleic acids depends on the length of the nucleic acids and the degree of complementation, variables well known in the art. The greater the degree of similarity or homology between two nucleotide sequences, the greater the value of T m for hybrids of nucleic acids having those sequences.
  • RNA:RNA, DNA:RNA, DNA:DNA The relative stability (corresponding to higher T m ) of nucleic acid hybridizations decreases in the following order: RNA:RNA, DNA:RNA, DNA:DNA.
  • equations for calculating T m have been derived (see Sambrook et al, 1989, 9.50-9.51).
  • the position of mismatches becomes more important, and the length of the oligonucleotide determines its specificity (see Sambrook et al, 1989, 11.7-11.8).
  • a minimum length for a hybridizable nucleic acid is at least about 10 nucleotides; preferably at least about 15 nucleotides; and more preferably the length is at least about 20 nucleotides.
  • standard hybridization conditions refers to a T m of 55 °C, and utilizes conditions as set forth above.
  • the T m is 60°C; in a more preferred embodiment, the T m is 65 °C.
  • “high stringency” refers to hybridization and/or washing conditions at 68 °C in 0.2XSSC, at 42 °C in 50% formamide, 4XSSC, or under conditions that afford levels of hybridization equivalent to those observed under either of these two conditions.
  • oligonucleotide refers to a nucleic acid, generally of at least 10, preferably at least 15, and more preferably at least 20 nucleotides, that is hybridizable to a genomic DNA molecule, a cDNA molecule, or an mRNA molecule encoding a gene, mRNA, cDNA, or other nucleic acid of interest. Oligonucleotides can be labeled, e.g., with 32 P-nucleotides or nucleotides to which a ligand molecule, such as biotin, has been covalently conjugated.
  • a labeled oligonucleotide can be used as a probe to detect the presence of a nucleic acid.
  • oligonucleotides (one or both of which may be labeled) can be used as PCR or sequencing primers, either for cloning full length or a fragment of CASP8, or to detect the presence of nucleic acids encoding CASP8.
  • an oligonucleotide of the invention can form a triple helix with a CASP8 DNA molecule.
  • a library of oligonucleotides arranged on a solid support can be used to detect various CASP8 polymo ⁇ hisms of interest.
  • oligonucleotides are prepared synthetically, preferably on a nucleic acid synthesizer. Accordingly, oligonucleotides can be prepared with non-naturally occurring phosphoester analog bonds, such as thioester bonds, etc.
  • homologous in all its grammatical forms and spelling variations refers to the relationship between proteins that possess a "common evolutionary origin,” including proteins from superfamilies (e.g., the immunoglobulin superfamily) and homologous proteins from different species (e.g., myosin light chain, etc.) (Reeck et al, Cell, 1987, 50:667). Such proteins (and their encoding genes) have sequence homology, as reflected by their high degree of sequence similarity.
  • sequence similarity in all its grammatical forms refers to the degree of identity or correspondence between nucleic acid or amino acid sequences of proteins that may or may not share a common evolutionary origin (see Reeck et al, supra).
  • sequence similarity when modified with an adverb such as "highly,” may refer to sequence similarity and may or may not relate to a common evolutionary origin.
  • two DNA sequences are "substantially homologous” or “substantially similar” when at least about 30%, and preferably at least about 50%, of the nucleotides match over the defined length of the DNA sequences.
  • sequences that are substantially homologous can be identified by comparing the sequences using standard software available in sequence data banks or from commercial sources (BLAST, DNA Strider, DNA Star, FASTA, etc.) using standard or default parameters, or in a Southern hybridization experiment under, for example, stringent conditions as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See, e.g., Sambrook et al, 1989; DNA Cloning, Vols. I & II, supra; Nucleic Acid Hybridization, supra.
  • two amino acid sequences are "substantially homologous" or “substantially similar” when greater than about 80%, preferably greater than about 90%, of the amino acids are identical, or greater than about 85%, preferably greater than about 95%, are similar (functionally identical).
  • the similar or homologous sequences are identified by alignment using, for example, the GCG (Genetics Computer Group, Program Manual for the GCG Package, Version 7, Madison, Wisconsin) pileup program, or any of the sequence alignment programs described above.
  • a gene encoding CASP8, whether genomic DNA or cDNA, can be isolated from any source, particularly from a human cDNA or genomic library. Methods for obtaining CASP8 gene are well known in the art, as described above (.see, e.g., Sambrook et al, 1989).
  • the DNA may be obtained by standard procedures known in the art from cloned DNA (e.g., a DNA "library”), and preferably is obtained from a cDNA library prepared from tissues with high level expression of the protein (e.g., a peripheral blood leukocyte cell library, since these are the cells that evidence high levels of expression of CASP8), by chemical synthesis, by cDNA cloning, or by the cloning of genomic DNA, or fragments thereof, purified from the desired cell (see, e.g., Sambrook et al, 1989; DNA cloning, supra).
  • clones derived from genomic DNA e.g., a DNA "library”
  • DNA may contain regulatory and intron DNA regions in addition to coding regions; clones derived from cDNA will not contain intron sequences. Whatever the source, the gene should be molecularly cloned into a suitable vector for propagation of the gene. Identification of the specific DNA fragment containing the desired CASP8 gene may be accomplished in a number of ways. For example, a portion of a CASP8 gene exemplified infra can be purified and labeled to prepare a labeled probe, and the generated DNA may be screened by nucleic acid hybridization to the labeled probe (Benton and Davis, Science, 1977, 196:180; Grunstein and Hogness, Proc. Natl. Acad. Sci. USA, 1975, 72:3961).
  • Those DNA fragments with substantial homology to the probe will hybridize.
  • highest stringency hybridization conditions are used to identify a homologous CASP8 gene. Further selection can be carried out on the basis of the properties of the gene, e.g., if the gene encodes a protein product having the isoelectric, electrophoretic, amino acid composition, partial or complete amino acid sequence, antibody binding activity, or ligand binding profile of CASP8 protein as disclosed herein. Thus, the presence of the gene may be detected by assays based on the physical, chemical, immunological, or functional properties of its expressed product.
  • the present invention also relates to cloning vectors containing genes encoding analogs and derivatives of CASP8 of the invention, that have the same or homologous functional activity as CASP8.
  • the production and use of derivatives and analogs related to CASP8 are within the scope of the present invention.
  • the derivative or analog is functionally active, i.e., capable of exhibiting one or more functional activities associated with a full-length, wild-type CASP8 of the invention.
  • Such functions include FADD and death receptor binding, induction of apoptosis, autoproteolytic cleavage, and aggregate formation.
  • Chimeric fusion proteins with CASP8, such as GST or HIS-tagged CASP8, are contemplated, as are fusion proteins that contain functional domains (discussed below).
  • CASP8 derivatives can be made by altering encoding nucleic acid sequences by substitutions, additions or deletions that provide for functionally equivalent molecules.
  • derivatives are made that have enhanced or increased functional activity relative to native CASP8.
  • chimeric derivatives with a greater self-aggregation potential e.g., prepared by inco ⁇ orating an additional or stronger aggregation sequence, may have increased functional activity.
  • a chimeric CASP8 protein that contains the FADD death domain may be especially effective for generation of a death-inducing signaling complex, even in the absence of FADD.
  • nucleotide coding sequences which encode substantially the same amino acid sequence as a CASP8 gene may be used in the practice of the present invention.
  • these include but are not limited to allelic genes and nucleotide sequences comprising all or portions of CASP8 genes which are altered by the substitution of different codons that encode the same amino acid residue within the sequence, thus producing a silent change.
  • the CASP8 derivatives of the invention include, but are not limited to, those containing, as a primary amino acid sequence, all or part of the amino acid sequence of a CASP8 protein including altered sequences in which functionally equivalent amino acid residues are substituted for residues within the sequence resulting in a conservative amino acid substitution.
  • one or more amino acid residues within the sequence can be substituted by another amino acid of a similar polarity and, if present, charge, which acts as a functional equivalent, resulting in a silent alteration.
  • Substitutes for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs.
  • the nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine.
  • Amino acids containing aromatic ring structures are phenylalanine, tryptophan, and tyrosine.
  • the polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine.
  • the positively charged (basic) amino acids include arginine, lysine and histidine.
  • the negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Such alterations will not be expected to affect apparent molecular weight as determined by polyacrylamide gel electrophoresis, or isoelectric point. Particularly preferred substitutions are:
  • genes encoding CASP8 derivatives and analogs of the invention can be produced by various methods known in the art.
  • the sequence can be cleaved at appropriate sites with restriction endonuclease(s), followed by further enzymatic modification if desired, isolated, and ligated in vitro.
  • the CASP8-encoding nucleic acid sequence can be mutated in vitro or in vivo, to create and/or destroy translation, initiation, and/or termination sequences, or to create variations in coding regions and/or form new restriction endonuclease sites or destroy preexisting ones, to facilitate further in vitro modification.
  • Any technique for mutagenesis known in the art can be used, including but not limited to, in vitro site-directed mutagenesis (Hutchinson, C, et al, J. Biol. Chem., 1978, 253:6551; Zoller and Smith, DNA, 1984, 3:479-488; Oliphant et al, Gene, 1986, 44:177; Hutchinson et al, Proc. Natl.
  • the identified and isolated gene can then be inserted into an appropriate cloning vector.
  • vector-host systems known in the art may be used. Possible vectors include, but are not limited to, plasmids or modified viruses, but the vector system must be compatible with the host cell used. Examples of vectors include, but are not limited to, E. coli, bacteriophages such as lambda derivatives, or plasmids such as pBR322 derivatives or pUC plasmid derivatives, e.g., pGEX vectors, pmal-c, pFLAG, etc.
  • the insertion into a cloning vector can, for example, be accomplished by ligating the DNA fragment into a cloning vector which has complementary cohesive termini.
  • the ends of the DNA molecules may be enzymatically modified.
  • any site desired may be produced by ligating nucleotide sequences (linkers) onto the DNA termini; these ligated linkers may comprise specific chemically synthesized oligonucleotides encoding restriction endonuclease recognition sequences.
  • Recombinant molecules can be introduced into host cells via transformation, transfection, infection, electroporation, etc., so that many copies of the gene sequence are generated.
  • the cloned gene is contained on a shuttle vector plasmid, which provides for expansion in a cloning cell, e.g., E. coli, and facile purification for subsequent insertion into an appropriate expression cell line, if such is desired.
  • a shuttle vector which is a vector that can replicate in more than one type of organism, can be prepared for replication in both E. coli and Saccharomyces cerevisiae by linking sequences from an E. coli plasmid with sequences from the yeast 2 ⁇ plasmid.
  • the nucleotide sequence coding for CASP8, including derivatives or analogs thereof, or a functionally active chimeric protein thereof can be inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for the transcription and translation of the inserted protein-coding sequence in vivo.
  • an appropriate expression vector i.e., a vector which contains the necessary elements for the transcription and translation of the inserted protein-coding sequence in vivo.
  • the nucleic acid encoding CASP8 of the invention is operationally associated with a promoter in an expression vector of the invention. Both cDNA and genomic sequences can be cloned and expressed under control of such regulatory sequences.
  • An expression vector also preferably includes a replication origin.
  • an CASP8 polypeptide of the invention can be prepared using well-known techniques in peptide synthesis, including solid phase synthesis (using, e.g., BOC of FMOC chemistry), or peptide condensation techniques.
  • polypeptide and “protein” may be used interchangeably to refer to the gene product (or corresponding synthetic product) of a CASP8 gene.
  • protein may also refer specifically to the polypeptide as expressed in cells.
  • a peptide is generally a fragment of a polypeptide, e.g., of about six or more amino acid residues.
  • the necessary transcriptional and translational signals can be provided on a recombinant expression vector, or they may be supplied by the native gene encoding CASP8 and/or its flanking regions.
  • control sequences whether a promoter or enhancer, or both, permit high level expression of caspase-8 in the target cell, particularly for gene therapy (described infra).
  • Potential host-vector systems include but are not limited to mammalian cell systems infected with virus (e.g., vaccinia virus, adeno virus, adeno-associated virus, he ⁇ es virus, etc.); insect cell systems infected with virus (e.g., baculovirus); microorganisms such as yeast containing yeast vectors; or bacteria transformed with bacteriophage, DNA, plasmid DNA, or cosmid DNA.
  • virus e.g., vaccinia virus, adeno virus, adeno-associated virus, he ⁇ es virus, etc.
  • insect cell systems infected with virus e.g., baculovirus
  • microorganisms such as yeast containing yeast vectors
  • bacteria transformed with bacteriophage DNA, plasmid DNA, or cosmid DNA.
  • the expression elements of vectors vary in their strengths and specificities. Depending on the host-vector system utilized, any one of a number of suitable transcription and translation elements
  • a recombinant CASP8 protein of the invention, or functional fragment, derivative, chimeric construct, or analog thereof, may be expressed chromosomally, after integration of the coding sequence by recombination.
  • any of a number of amplification systems may be used to achieve high levels of stable gene expression (See Sambrook et al, 1989).
  • any of the methods previously described for the insertion of DNA fragments into a cloning vector may be used to construct expression vectors containing a gene consisting of appropriate transcriptional/translational control signals and the protein coding sequences. These methods may include in vitro recombinant DNA and synthetic techniques and in vivo recombination (genetic recombination). Expression of CASP8 protein may be controlled by any promoter/enhancer element known in the art, but these regulatory elements must be functional in the host selected for expression. In one embodiment, the promoter permits high level expression in a mammalian, and more preferably human, host cell. For example, viral promoters, some of which are listed below, often permit high level expression. Other such promoters are known.
  • Promoters which may be used to control CASP8 gene expression include, but are not limited to, cytomegalo virus (CMV) promoter (U.S. Patent Nos. 5,168,062 and 5,385,839), the SV40 early promoter region (Benoist and Chambon, Nature, 1981, 290:304-310), the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto, et al, Cell, 1980, 22:787-797), the he ⁇ es thymidine kinase promoter (Wagner et al, Proc. Natl. Acad. Sci.
  • CMV cytomegalo virus
  • alpha 1- antitrypsin gene control region which is active in the liver (Kelsey et al, Genes and Devel., 1987, 1:161-171), beta-globin gene control region which is active in myeloid cells (Mogram et al, Nature, 1985, 315:338-340; Kollias et al, Cell, 1986, 46:89-94), myelin basic protein gene control region which is active in oligodendrocyte cells in the brain (Readhead et al, Cell, 1987, 48:703-712), myosin light chain-2 gene control region which is active in skeletal muscle (Sani, Nature, 1985, 314:283-286), and gonadotropic releasing hormone gene control region which is active in the hypothalamus (Mason et al, Science, 1986, 234:1372-1378).
  • promoters useful for practice of this invention are ubiquitous promoters (e.g., HPRT, vimentin, actin, tubulin), intermediate filament promoters (e.g., desmin, neuro filaments, keratin, GFAP), therapeutic gene promoters (e.g., MDR type, CFTR, factor VIII), tissue- specific promoters (e.g., actin promoter in smooth muscle cells), promoters which are preferentially activated in dividing cells, promoters which respond to a stimulus (e.g., steroid hormone receptor, retinoic acid receptor), tetracycline-regulated transcriptional modulators, retro viral LTR, metallothionein, El a, and MLP promoters.
  • ubiquitous promoters e.g., HPRT, vimentin, actin, tubulin
  • intermediate filament promoters e.g., desmin, neuro filaments, keratin, GFAP
  • therapeutic gene promoters e.g., MDR type,
  • promoters specific for expression in cells of the CNS can be used, e.g., for gene therapy of neuroblastoma or another CNS tumor.
  • the promoters for the human tyrosine hydroxylase gene Kim et al, Nucl. Acids Res., 1998, 26:1793-800
  • human dopamine beta-hydroxylase (DBH) gene Zellmer et al, J. of Neurosci., 1995, 15:8109-20
  • human ENC-1 gene Hernandez et al, Exp. Cell Res., 1998, 242:470-7) are CNS-specific.
  • Vectors are introduced into the desired host cells by methods known in the art, e.g., transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, lipofection (liposome fusion), use of a gene gun, or a DNA vector transporter (see, e.g., Wu et al, J. Biol. Chem., 1992, 267:963-967; Wu and Wu, J. Biol. Chem., 1988, 263:14621-14624; Canadian Patent Application No. 2,012,311).
  • CASP8 polypeptides whether produced recombinantly or by chemical synthesis or purified from cells that express the protein endogenously, and fragments or other derivatives or analogs thereof, including fusion proteins, may be used as an immunogen to generate antibodies that recognize the CASP8 polypeptide.
  • Such antibodies include but are not limited to polyclonal, monoclonal, chimeric, single chain, Fab fragments, and an Fab expression library. Such an antibody is specific for human CASP8.
  • CASP8 polypeptide or derivative or analog thereof various procedures known in the art may be used for the production of polyclonal antibodies to CASP8 polypeptide or derivative or analog thereof.
  • various host animals can be immunized by injection with the CASP8 polypeptide, or a derivative (e.g., fragment or fusion protein) thereof, including but not limited to rabbits, mice, rats, sheep, goats, etc.
  • the CASP8 polypeptide or fragment thereof can be conjugated to an immunogenic carrier, e.g., bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH).
  • BSA bovine serum albumin
  • KLH keyhole limpet hemocyanin
  • adjuvants may be used to increase the immunological response, depending on the host species, including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Gueri ⁇ ) and Corynebacterium parvum.
  • BCG Bacille Calmette-Gueri ⁇
  • Corynebacterium parvum bacille Calmette-Gueri ⁇
  • any technique that provides for the production of antibody molecules by continuous cell lines in culture may be used. These include but are not limited to the hybridoma technique originally developed by Kohler and Milstein (Nature, 1975, 256:495-497), as well as the trioma technique, the human B-cell hybridoma technique (Kozbor et al, Immunology Today,
  • such fragments include but are not limited to: the F(ab') 2 fragment which can be produced by pepsin digestion of the antibody molecule; the Fab' fragments which can be generated by reducing the disulfide bridges of the F(ab') 2 fragment, and the Fab fragments which can be generated by treating the antibody molecule with papain and a reducing agent.
  • screening for the desired antibody can be accomplished by techniques known in the art, e.g., radioimmunoassay, ELISA (enzyme-linked immunosorbant assay), "sandwich” immunoassays, immunoradiometric assays, gel diffusion precipitin reactions, immunodiffusion assays, in situ immunoassays (using colloidal gold, enzyme or radioisotope labels, for example), western blots, precipitation reactions, agglutination assays (e.g., gel agglutination assays, hemagglutination assays), complement fixation assays, immunofluorescence assays, protein A assays, and immunoelectrophoresis assays, etc.
  • radioimmunoassay e.g., ELISA (enzyme-linked immunosorbant assay), "sandwich” immunoassays, immunoradiometric assays, gel diffusion precipitin reactions, immunodiffusion assays, in situ immunoa
  • antibody binding is detected by detecting a label on the primary antibody.
  • the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody.
  • the secondary antibody is labeled.
  • Many means are known in the art for detecting binding in an immunoassay and are within the scope of the present invention. For example, to select antibodies which recognize a specific epitope of an CASP8 polypeptide, one may assay generated hybridomas for a product which binds to an CASP8 polypeptide fragment containing such epitope.
  • the foregoing antibodies can be used in methods known in the art relating to the localization and activity of the CASP8 polypeptide, e.g., for Western blotting, imaging CASP8 polypeptide in situ, measuring levels thereof in appropriate physiological samples, etc. using any of the detection techniques mentioned above or known in the art. Such antibodies may exhibit su ⁇ risingly superior properties in immunodiagnostic (or immunoprognostic) assays for CASP8. Diagnostics
  • the present invention provides for evaluating cancer in a subject or patient based on detecting whether caspase-8 has been inactivated. This evaluation can provide either a diagnosis or a prognosis, or both. For example, detecting inactivation of caspase-8 in neuroblastoma provides a diagnosis of aggressive neuroblastoma, and a prognosis of a poor outcome from traditional therapies.
  • diagnosis in any grammatical form refers to the identification of a particular disease condition in a subject or patient. As the skilled physician knows, almost any diagnosis is based on a multiple of determinants, including symptomology, histology, and other criteria, which together form a diagnosis. Thus, when used herein, diagnosis according to the invention is one component or determinant of the final diagnosis.
  • diagnosis in any grammatical form refers to prediction of a disease outcome, e.g., whether the subject suffering from the disease is likely to improve or regress. Any form of cancer, e.g., as discussed above, can be evaluated using the diagnostic methods of the invention. Preferably, the cancer is neuroblastoma (particularly juvenile neuroblastoma), small-cell lung carcinoma, non-small-cell lung carcinoma, colorectal carcinoma, or uterine cervical carcinoma.
  • kits contain, at least, a detection assay for inactivation of caspase-8.
  • Nucleic acid assays for inactivation of CASP8 are based on detection of mutations or modifications in the CASP8 gene that result in its inactivation.
  • the DNA may be obtained from any cell source.
  • Non-limiting examples of cell sources available in clinical practice include without limitation blood cells, buccal cells, cervicovaginal cells, epithelial cells from urine, fetal cells, or any cells present in tissue obtained by biopsy.
  • Cells may also be obtained from body fluids, including without limitation blood, plasma, serum, lymph, milk, cerebrospinal fluid, saliva, sweat, urine, feces, and tissue exudates (e.g., pus) at a site of infection or inflammation.
  • DNA is extracted from the cell source or body fluid using any of the numerous methods that are standard in the art.
  • the particular method used to extract DNA will depend on the nature of the source. Generally, the minimum amount of DNA to be extracted for use in the present invention is about 25 pg (corresponding to about 5 cell equivalents of a genome size of 4 x 10 9 base pairs).
  • Modifications of the CASP8 genomic DNA include genomic methylation of the promoter.
  • evaluation of inactivation of CASP8 involves an assay for methylation of the CASP8 promoter. It has been found that in most cases, inactivation of CASP8 results from methylation of the promoter and not from gene deletion (see Examples 2, 3, infra).
  • methylation of the promoter can be detected by a methylation polymerase chain reaction (PCR) assay (see Belinsky et al, Proc. Natl. Acad. Sci.
  • PCR methylation polymerase chain reaction
  • primer pairs can be used to detect methylated, bisulfite treated promoter DNA (e.g., SEQ ID NOS:29 and 30), unmethylated, bisulfite-treated promoter DNA (e.g., SEQ ID NOS:31 and 32), and wild-type (un-bisulfite treated) DNA (e.g., SEQ ID NOS:33 and 34).
  • SEQ ID NOS:29 and 30 primers designed based on the promoter regions depicted in SEQ ID NOS: 1 and 2.
  • unmethylated, bisulfite-treated promoter DNA e.g., SEQ ID NOS:31 and 32
  • wild-type (un-bisulfite treated) DNA e.g., SEQ ID NOS:33 and 34.
  • Mutations include an insertion in the gene, deletion of the gene, truncation of the gene (e.g., due to a nonsense, missense, or frameshift mutation), or disregulation of gene expression (e.g., due to a frameshift mutation or a splice-site mutation).
  • CASP8 is heterozygously or homozygously deleted from chromosome 2. Identification of gene deletion is readily accomplished using nucleic acid probes or PCR analysis.
  • Determination of polymo ⁇ hic positions is achieved by any means known in the art, including but not limited to direct sequencing, hybridization with allele-specific oligonucleotides, allele- specific PCR, ligase-PCR, HOT cleavage, denaturing gradient gel electrophoresis (DGGE), and single-stranded conformational polymo ⁇ hism (SSCP).
  • Direct sequencing may be accomplished by any method, including without limitation chemical sequencing, using the Maxam-Gilbert method; by enzymatic sequencing, using the Sanger method; mass spectrometry sequencing; and sequencing using a chip-based technology (see, e.g., Little et al, Genet. Anal., 1996, 6:151).
  • DNA from a subject is first subjected to amplification by polymerase chain reaction (PCR) using specific amplification primers.
  • PCR polymerase chain reaction
  • Gene expression, or lack of gene expression, can be directly evaluated by detecting CASP8 mRNA.
  • Methods for detecting mRNA include Northern blotting and reverse transcriptase (RT)-PCR. These methods can be used to determine whether or not expression occurs, and whether a truncated (or oversized) message is expressed. All of these factors can help establish inactivation of caspase-8.
  • a nucleic acid assay kit of the invention will comprise a nucleic acid that specifically hybridizes under stringent conditions to a CASP8 gene, and an assay detector, e.g., a label.
  • a primer pair will be included; in this case, the detector may simply be a reagent such as ethidium bromide to quantify amplified DNA.
  • a nucleic acid based kit of the invention includes primer pairs for PCR analysis of CASP8 promoter methylation.
  • the kit contains primer pairs that can be used to detect methylated, bisulfite treated promoter DNA (e.g., SEQ ID NOS:29 and 30), unmethylated, bisulfite-treated promoter DNA (e.g., SEQ ID NOS:31 and 32), and wild-type (un-bisulfite treated) DNA (e.g., SEQ ID NOS:33 and 34).
  • Optional components include buffer or buffer reagents, nucleotides, and instructions for use of the kit. If possible, a positive control is also included, e.g., a probe or primer pair for an endogenously expressed gene, such as actin or tubulin.
  • Protein Based Assays As an alternative to analyzing CASP8 nucleic acids, one can evaluate caspase-8 on the basis of protein expression. Indeed, this assay may be the most informative, since CASP8 mRNA levels may appear high, but a mutation in the sequence may make the mRNA less effective for translation, resulting in reduction or elimination of protein expression.
  • caspase-8 is detected by immunoassay.
  • immunoassay for example, as exemplified infra, Western blotting permits detection of the presence or absence of caspase-8.
  • Other immunoassay formats e.g., as discussed above in connection with CASP8-specific antibodies, can also be used in place of Western blotting.
  • a biochemical assay can be used to detect expression of caspase-8, e.g., by the presence or absence of a band by polyacrylamide gel electrophoresis; by the presence or absence of a chromatographic peak by any of the various methods of high performance liquid chromatography, including reverse phase, ion exchange, and gel permeation; by the presence or absence of caspase-8 in analytical capillary electrophoresis chromatography, or any other quantitative or qualitative biochemical technique known in the art.
  • biopsy tissue is obtained from a subject.
  • the tumor cells should be purified from other tissue to ensure that contaminating CASP8 from normal cells is not detected.
  • Antibodies that are capable of binding to caspase-8 are then contacted with samples of the tissue under conditions that permit antibody binding to determine the presence or absence of caspase-8.
  • antibodies that distinguish polymo ⁇ hic variants of caspase-8 can be used.
  • the antibodies may be polyclonal or monoclonal, preferably monoclonal. Measurement of specific antibody binding to cells may be accomplished by any known method, e.g., quantitative flow cytometry, or enzyme-linked or fluorescence-linked immunoassay.
  • the presence or absence of a particular mutation, and its allelic distribution is determined by comparing the values obtained from a patient with norms established from populations of patients having known polymo ⁇ hic patterns.
  • kits of the invention provides a caspase-8 detector, e.g., a detectable antibody (which may be directly labeled or which may be detected with a secondary labeled reagent).
  • a caspase-8 detector e.g., a detectable antibody (which may be directly labeled or which may be detected with a secondary labeled reagent).
  • anti-tumor gene therapy refers to a gene therapy targeted to cells of a tumor, i.e., cancer, which causes tumor necrosis, apoptosis, growth regulation, i.e., regression or suppression of the tumor.
  • anti-tumor gene therapy refers to administration or delivery of a gene encoding caspase-8, either alone or in combination with other genes effective for treating tumors.
  • anti -tumor gene therapies of the prior art include, but are by no means limited to, introduction of a suicide gene; introduction of an apoptosis gene; introduction of a tumor suppresser gene; and introduction of an oncogene antagonist gene.
  • anti-tumor genes such as CASP8
  • CASP8 are supplemented with immunostimulatory genes to enhance recruitment and activation of immune effector cells.
  • a viral, such as adenovirus, vector is used (see, e.g., PCT Publication No. WO 95/14101), the presence of adenoviral antigens could also provide an adjuvant effect to overall enhanced immune responsiveness.
  • Gene therapy refers to transfer of a gene encoding an effector molecule into cells, in this case of the tumor.
  • Gene therapy vectors include, but are not limited to, viral vectors (including retroviruses and DNA viruses), naked DNA vectors, and DNA-transfection agent admixtures.
  • a therapeutically effective amount of the vectors are delivered in a pharmaceutically acceptable carrier.
  • the phrase "therapeutically effective amount” is used herein to mean an amount sufficient to reduce by at least about 15 percent, preferably by at least 50 percent, more preferably by at least 90 percent, and most preferably prevent, a clinically significant deficit in the activity, function and response of the host, e.g., tumor progression, metastasises, or progression to the next stage of cancer.
  • a therapeutically effective amount is sufficient to cause an improvement in a clinically significant condition in the host, e.g., to induce remission, reduce tumor size or burden, or both, or increase the time from treatment until relapse.
  • pharmaceutically acceptable refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human.
  • the term "pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
  • carrier refers to a diluent, adjuvant, excipient, or vehicle with which the compound is administered.
  • Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like.
  • Water or aqueous solution saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions.
  • Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences” by E.W. Martin. Such methods, including routes of administration and dose, are well known in the art. These are discussed in greater detail in a section directed to "Gene Therapy Vectors" below, as well as in the references disclosed therein.
  • Gene therapy in accordance with the invention can be used to treat any cancer, but particularly tumors with an amplified MYC oncogene.
  • caspase- 8 is inactivated in the tumor cells of the cancer.
  • increasing the level of expression of caspase-8 beyond endogenous levels is expected to initiate cell death-inducing signaling.
  • modified forms of caspase-8 discussed supra
  • enhanced death-inducing activity can be used in tumor cells that express wild-type caspase-8 endogenously.
  • assays that detect expression e.g., Northern assays
  • translation e.g., immunoassays
  • CASP8 may not differentiate a defective gene product from wild- type, thus delivery of the wild-type gene may be useful even if it appears that the cell expresses caspase-8.
  • caspase-8 gene therapy of a tumor can be combined with other anti-tumor therapies, including but by no means limited to suicide gene therapy, anti-oncogene or tumor suppressor gene therapy, administration of tumor growth inhibitors, administration of angiogenesis inhibitors, immune therapies with an immunologically active polypeptide (including immunostimulation, e.g., in which the active polypeptide is a cytokine, lymphokine, or chemokine, and vaccination, in which the active polypeptide is a tumor specific or tumor associated antigen), and conventional cancer therapies (chemotherapy and radiation therapy).
  • immunologically active polypeptide including immunostimulation, e.g., in which the active polypeptide is a cytokine, lymphokine, or chemokine
  • vaccination in which the active polypeptide is a tumor specific or tumor associated antigen
  • conventional cancer therapies chemotherapy and radiation therapy
  • Suicide gene therapies Introduction of genes that encode enzymes capable of conferring to tumor cells sensitivity to chemotherapeutic agents (suicide gene) has proven to be an effective anti-tumor gene therapy.
  • a representative example of such a suicide gene is thymidine kinase of he ⁇ es simplex virus. Additional examples are thymidine kinase of varicella zoster virus and the bacterial gene cytosine deaminase which can convert 5-fluorocytosine to the highly toxic compound 5- fluorouracil.
  • the prodrug useful in the methods of the present invention is any that can be converted to a toxic product, i.e., toxic to tumor cells.
  • the prodrug is converted to a toxic product by the gene product of the therapeutic nucleic acid sequence in the vector useful in the method of the present invention.
  • a prodrug is ganciclovir, which is converted in vivo to a toxic compound by HSV-tk.
  • Other representative examples of pro-drugs include acyclovir, FIAU [l-(2-deoxy-2- fluoro- ⁇ -D-arabinofuranosyl)-5-iodouracil], 6-methoxypurine arabino-side for VZV-tk, and 5-fiuorocytosine for cytosine deambinase.
  • Anti-oncogene and tumor suppresser gene therapies Tumor initiation and progression in many cancer types are linked to mutations in oncogenes (e.g., ras, myc) and tumor suppresser genes (e.g., retinoblastoma protein, ⁇ 53).
  • oncogenes e.g., ras, myc
  • tumor suppresser genes e.g., retinoblastoma protein, ⁇ 53.
  • a number of approaches are being pursued using anti-oncogene molecules including monoclonal antibodies, single chain antibody vectors, antisense oligonucleotide constructs, ribozymes and immunogenic peptides (Chen, Mol. Med. Today, 1997, 3:160-167; Spitz, et al, Anticancer Res., 1996, 16:3415-3422; Indolfi et al, Nat. Med., 1996,
  • a vector is any means for the transfer of a nucleic acid according to the invention into a host cell.
  • Prefe ⁇ ed vectors for transient expression are viral vectors, such as retroviruses, he ⁇ es viruses, adenoviruses and adeno-associated viruses.
  • viral vectors such as retroviruses, he ⁇ es viruses, adenoviruses and adeno-associated viruses.
  • a gene encoding a functional caspase-8 protein or polypeptide domain fragment thereof can be introduced in vivo, ex vivo, or in vitro using a viral vector or through direct introduction of DNA.
  • Expression in targeted tissues can be effected by targeting the transgenic vector to specific cells, such as with a viral vector or a receptor ligand, or by using a tissue-specific promoter, or both. Targeted gene delivery is described in PCT Publication No. WO 95/28494.
  • Viral vectors are DNA-based vectors and retroviral vectors. Methods for constructing and using viral vectors are known in the art (see, e.g., Miller and Rosman, BioTechniques, 1992, 7:980-990).
  • the viral vectors are replication defective, that is, they are unable to replicate autonomously in the target cell.
  • the genome of the replication defective viral vectors which are used within the scope of the present invention lack at least one region which is necessary for the replication of the virus in the infected cell. These regions can either be eliminated (in whole or in part), be rendered non- functional by any technique known to a person skilled in the art.
  • These techniques include the total removal, substitution (by other sequences, in particular by the inserted nucleic acid), partial deletion or addition of one or more bases to an essential (for replication) region.
  • Such techniques may be performed in vitro (on the isolated DNA) or in situ, using the techniques of genetic manipulation or by treatment with mutagenic agents.
  • the replication defective virus retains the sequences of its genome which are necessary for encapsidating the viral particles.
  • DNA viral vectors include an attenuated or defective DNA virus, such as but not limited to he ⁇ es simplex virus (HSV), papillomavirus, Epstein Barr virus (EBV), adenovirus, adeno-associated virus (AAV), and the like.
  • HSV he ⁇ es simplex virus
  • EBV Epstein Barr virus
  • AAV adeno-associated virus
  • Defective viruses which entirely or almost entirely lack viral genes, are preferred. Defective virus is not infective after introduction into a cell.
  • Use of defective viral vectors allows for administration to cells in a specific, localized area, without concern that the vector can infect other cells. Thus, a specific tissue can be specifically targeted.
  • particular vectors include, but are not limited to, a defective he ⁇ es virus 1 (HSV1) vector (Kaplitt et al, Molec. Cell.
  • a defective he ⁇ es simplex virus has been shown to be effective for delivery of genes, particularly to cells of the CNS (see, e.g., Belloni et al, Human Gene Therapy, 1996, 7:2015-24).
  • Recombinant defective adenoviruses have been used for transferring foreign genes into cells, particularly for gene therapy of tumors (as noted above), and for delivery of therapeutic genes to cells of the central nervous system.
  • PCT Publication Nos.WO 94/08026 and WO 94/08026 describe recombinant adeno virus vectors for the transfer of foreign genes into the central nervous system (CNS).
  • Other examples of gene delivery to the CNS include the following: French
  • FR2717824 discloses adenoviruses containing DNA from glial derived neutrophilic factors, which infected nerve cells very efficiently; various publications describe adenoviral vectors that express glial maturation factor (FR2717497), brain derived neurotropic factor (FR2717496) and acidic fibroblast growth factor (FR2717495); PCT Publication No. WO 95/26409 describes adenoviruses containing the DNA sequence for basic fibroblast growth factor to infect cells directly or via implants to treat neurological disorders; PCT Publication No.
  • WO 96/00790 describes adenoviruses containing DNA encoding superoxide dismutase (SOD) to treat neurodegenerative diseases and excessive SOD expression; and PCT Publication No. WO 96/01902 describes adenoviruses expressing nitric oxide synthase for gene therapy where angiogenesis is required for treating disorders of the CNS.
  • SOD superoxide dismutase
  • Adenoviruses can be genetically modified to reduce the levels of viral gene transcription and expression, including adenoviruses defective in the El and E4 regions (PCT Publication No. WO 96/22378) and adenoviruses with an inactivated El region but also with altered genomic organization reducing the number of viable viral particles produced if recombination occurs with the host genome (PCT Publication No. WO 96/13596).
  • PCT Publication No. WO 96/10088 describes defective adenoviruses with an inactivated EVa2 gene.
  • PCT Publication No. WO 95/02697 describes an adeno virus defective in regions El and E2, E4, or L1-L5.
  • plasmovirus Combination virus
  • a gene can be introduced using a combined virus, also termed plasmovirus (Genopoietic, France) vector system.
  • Plasmovirus systems permit one cycle of infectious virus formation in infected host cells.
  • a complementing gene(s) for defective viral genome sequences and the defective viral sequences are both provided to target cells in vivo or in vitro.
  • the primary infected cells produce infectious, defective virus.
  • plasmovirus technology amplifies gene delivery in vitro and, particularly, in vivo.
  • Non-viral vectors can be introduced in vivo by lipofection, as naked DNA, or with other transfection facilitating agents (peptides, polymers, etc.).
  • Synthetic cationic lipids can be used to prepare liposomes for in vivo transfection of a gene encoding a marker (Feigner et. al, Proc. Natl. Acad. Sci. USA, 1987, 84:7413-7417; Feigner and Ringold, Science, 1989, 337:387-388; see Mackey et al, Proc. Natl. Acad. Sci. USA, 1988, 85:8027-8031; Ulmer et al, Science, 1993, 259:1745-1748).
  • lipid compounds and compositions for transfer of nucleic acids are described in PCT Publication Nos. WO 95/18863 and WO 96/17823, and in U.S. Patent No. 5,459,127.
  • Lipids may be chemically coupled to other molecules for the pu ⁇ ose of targeting (see Mackey et. al, supra).
  • Targeted peptides e.g., hormones or neurotransmitters, and proteins such as antibodies, or non-peptide molecules could be coupled to liposomes chemically.
  • a nucleic acid in vivo, is also useful for facilitating transfection of a nucleic acid in vivo, such as a cationic oligopeptide (e.g., PCT Publication No. WO 95/21931), peptides derived from DNA binding proteins (e.g., PCT Publication No. WO 96/25508), or a cationic polymer (e.g., PCT Publication No. WO 95/21931).
  • a cationic oligopeptide e.g., PCT Publication No. WO 95/21931
  • peptides derived from DNA binding proteins e.g., PCT Publication No. WO 96/25508
  • a cationic polymer e.g., PCT Publication No. WO 95/21931
  • naked DNA vectors for gene therapy can be introduced into the desired host cells by methods known in the art, e.g., transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, use of a gene gun, or use of a DNA vector transporter (see, e.g., Wu et al, J. Biol. Chem., 1992, 267:963-967; Wu and Wu, J. Biol. Chem., 1988, 263:14621-14624; Canadian Patent Application No. 2,012,311; Williams et al, Proc. Natl. Acad. Sci.
  • the present invention provides for further enhancement of the anti- tumor effect by including additional anti-tumor treatments with the anti-tumor gene.
  • additional anti-tumor treatments with the anti-tumor gene for example, the present invention contemplates further combinations with tumor growth inhibitors, anti-angiogenesis treatment, tumor antigen and whole tumor vaccines, chemotherapeutic agents, radiation, and surgery (tumor resection).
  • Tumor growth inhibitors are used herein to refer to a protein that inhibits tumor growth, such as but not limited to interferon (IFN)- ⁇ , tumor necrosis factor (TNF)- ⁇ , TNF- ⁇ , and similar cytokines.
  • a tumor growth inhibitor can be an antagonist of a tumor growth factor.
  • Such antagonists include, but are not limited to, antagonists of tumor growth factor (TGF)- ⁇ and IL-10.
  • TGF tumor growth factor
  • the present invention contemplates administration of tumor growth inhibitor proteins systemically, or alternatively by gene therapy. In a specific gene therapy embodiment, the gene therapy vector is administered directly to the tumor.
  • Anti-angiogenic factors Tumor angiogenesis is an integral part of tumor progression and a variety of therapies targeted to inhibit angiogenesis are under development as cancer therapies. Anti-angiogenesis molecules vary from anti- angiogenic proteins to small molecules that block growth factor receptor mediated effects. Anti-angiogenesis therapies primarily reverse the growth/apoptosis balance of the tumor and induce dormancy. Once the administration of these therapies is halted, angiogenesis can resume and tumor growth progresses.
  • An "anti-angiogenic factor” is a molecule that inhibits angiogenesis, particularly by blocking endothelial cell migration.
  • Such factors include fragments of angiogenic proteins that are inhibitory (such as the ATF of urokinase), angiogenesis inhibitory factors, such as angiostation (O'Reilly et al, Cell, 1994, 79:315-328) and endostatin; tissue inhibition of metalloproteinase (Johnson et al, J. Cell. Physiol., 1994, 160:194-202); soluble receptors of angiogenic factors, such as the urokinase receptor or FGF/VEGF receptor (Wilhem et al, FEBS Letters, 1994, 337:131-134); molecules which block endothelial cell growth factor receptors (O'Reilly et.
  • inhibitory such as the ATF of urokinase
  • angiogenesis inhibitory factors such as angiostation (O'Reilly et al, Cell, 1994, 79:315-328) and endostatin
  • tissue inhibition of metalloproteinase Johnson et al,
  • an anti-angiogenic factor for use in the invention is a protein or polypeptide, which may be encoded by a gene transfected into tumors using vectors of the invention.
  • the vectors of the invention can be used to deliver a gene encoding an anti-angiogenic protein into a tumor in accordance with the invention.
  • Immune activation Administration of various immunostimulatory molecules (cytokines, lymphokines, and chemokines, for example), such as CM-CSF and IL-2, can stimulate any immune response in conjunction with the tumor suppressor activity of caspase-8.
  • the immunostimulatory molecules can be delivered as proteins, e.g., by intravenous injection, or as therapeutic expression vectors, for expression in the host.
  • TAA tumor associated antigens
  • Chemotherapeutic agents are effective in inhibiting tumor growth and metastasis
  • the vectors and methods of the present invention are advantageously used with other treatment modalities, including without limitation surgery, radiation, chemotherapy, and other gene therapies.
  • the vectors of the invention can be administered in combination with nitric oxide inhibitors, which have vasoconstrictive activity and reduce blood flow to the tumor.
  • a vector of the invention can be administered with a chemotherapeutic such as, though not limited to, taxol, taxotere and other taxoids (e.g., as disclosed in U.S. Patent Nos. 4,857,653; 4,814,470; 4,924,011, 5,290,957; 5,292,921; 5,438,072; 5,587,493; European Patent No. EP 253 738; and PCT Publication Nos.
  • chemotherapeutic such as, though not limited to, taxol, taxotere and other taxoids (e.g., as disclosed in U.S. Patent Nos. 4,857,653; 4,814,470; 4,924,011, 5,290,957; 5,292,921; 5,438,072; 5,587,493; European Patent No. EP 253 738; and PCT Publication Nos.
  • WO 91/17976 WO 93/00928, WO 93/00929, and WO 96/01815), or other chemotherapeutics, such as cis-platin (and other platinum intercalating compounds), etoposide and etoposide phosphate, bleomycin, mitomycin C, CCNU, doxorubicin, daunorubicin, idarubicin, ifosfamide, and the like.
  • cis-platin platinum intercalating compounds
  • etoposide and etoposide phosphate bleomycin, mitomycin C, CCNU, doxorubicin, daunorubicin, idarubicin, ifosfamide, and the like.
  • CASP8 the role of CASP8 in cancer provides for development of screening assays, particularly for high throughput screening of molecules that agonize or antagonize the activity of CASP8.
  • indicator cells that are specially engineered to indicate the activity of CASP8, particularly initiation of the death process, can serve as targets to identify either a CASP8 inducer or replacement.
  • cells in which a CASP8 gene is inactivated are contacted with a candidate compound and cell apoptosis is evaluated or detected.
  • Such an assay will identify CASP8 agonists, i.e., molecules that re-activate CASP8 expression (such as 5-aza-2'-deoxycytidine), or substitutes, i.e., that activate the death pathway in the absence of CASP8.
  • CASP8 agonists i.e., molecules that re-activate CASP8 expression (such as 5-aza-2'-deoxycytidine), or substitutes, i.e., that activate the death pathway in the absence of CASP8.
  • cells in which CASP8 is inactivated because of methylation of the promoter are used in the assay.
  • Candidate compounds that lead to caspase-8 expression or initiation of apoptosis are selected for their ability to overcome the promoter methylation.
  • cells that express caspase-8 are contacted with a candidate compound and cell apoptosis is evaluated or detected.
  • Such an assay will indentify CASP8 antagonists, i.e., molecules that inhibit CASP8 activity and prevent initiation of the cell death pathway. Accordingly, the present invention contemplates methods for identifying specific agonists and antagonists of CASP8 activity using various screening assays known in the art.
  • Any screening technique known in the art can be used to screen for CASP8 agonists or antagonists.
  • the present invention contemplates screens for synthetic small molecules as well as screens for natural molecules that agonize or antagonize the activity of CASP8 in vivo.
  • natural products libraries can be screened using assays of the invention for molecules that agonize or antagonize CASP8 activity.
  • One approach uses recombinant bacteriophage to produce large libraries. Using the "phage method" (Scott and Smith, Science, 1990, 249:386-390; Cwirla, et al, Proc. Natl. Acad. Sci.
  • synthetic libraries (Needels et al, Proc. Natl. Acad. Sci. USA, 1993, 90:10700-4; Ohlmeyer et al, Proc. Natl. Acad. Sci. USA, 1993, 90:10922-10926; PCT Publication Nos. WO 92/00252 and WO 94/28028) and the like can be used to screen for CASP8 ligands according to the present invention.
  • Various reporter gene assays can be used to evaluate initiation of the death-inducing signaling complex.
  • a green fluorescent protein expression assay permits evaluation of caspase-8 activity.
  • GFP has been modified to produce proteins that remain functional but have different fluorescent properties, including different excitation and emission spectra (U.S. Patent No. 5,625,048 and PCT Publication No. WO 98/06737); an enzyme recognition site (PCT Publication No. WO 96/23898); increased intensity compared to the parent proteins (PCT Publication No. WO 97/11094); higher levels of expression in mammalian cells (PCT Publication No. WO 97/26633); twenty times greater fluorescence intensity than wild-type GFP (PCT Publication No. WO 97/42320); and mutants excitable with blue and white light (PCT Publication No. WO 98/21355).
  • reporter genes include luciferase, ⁇ -galactosidase ( ⁇ -gal or lac-Z), chloramphenicol transferase (CAT), horseradish peroxidase, and alkaline phosphatase.
  • CAT chloramphenicol transferase
  • Reporter gene expression can be tied to expression or activation of any component of the DISC system.
  • a GFP-expression vector containing the death receptor DR3 was used to evaluate caspase-8 activity.
  • Other DISC proteins, such as the Fas receptor, FADD, FasL, FAP, FAF, Trail/DR-2, TNFRp55, TRADD, RIP, and L32 can be similarly modified.
  • candidate compound can be tested for their ability to inhibit apoptosis in cells that express caspase-8, or for their ability to initiate apoptosis in cells in which caspace 8 is inactivated.
  • This Example describes isolation of the human gene corresponding to caspase-8, CASP8, and determination of its structure and chromosomal location.
  • the present study details this characterization, EcoRI and Hindlll restriction fragment length polymo ⁇ hisms (RFLPs), and demonstrates that CASP8 is localized to human chromosome 2 band q33-34, a region frequently deleted in human tumors. These results permit further study of the apoptotic signaling pathway in tumor cells.
  • Human caspase-8 cDNAs were obtained by computer analysis of a human EST library (Washington University-NCI Human EST Project). Three EST cDNAs were isolated. DNA sequencing of the caspase-8 EST DNAs was carried out as previously described using automated fluorescent-based DNA sequence analysis using Perkin Elmer/ Applied Biosystems Division DNA sequencers models 373-377, with TaqFS enzyme and multiple oligonucleotide primers derived from the published cDNA sequence (Muzio et al, Cell, 1996, 85:817; Boldin et al, Cell, 1996, 85:803). Oligonucleotides were spaced approximately 80-100 bp apart spanning the cDNA.
  • the human CASP8 gene was isolated from a human BAC genomic library (Genome Systems, Inc.) by hybridization with the co ⁇ esponding human cDNA. Two BAC clones containing CASP8 related DNA sequences were identified (clones BAC43 and BAC 44 with the clone addresses BACH 43(K10) and BACH 44(M7); Genome Systems, Inc., St. Louis, MO). Their identity was confirmed by DNA sequence analysis of the entire coding region, the 3' UTR, and a portion of the 5' untranslated region (UTR).
  • Hindlll fragments containing the entire gene were subcloned into pKS, and the resulting plasmid DNA used for double strand DNA sequence analysis.
  • Oligonucleotide primers designed for sequencing the cDNA clones all exons and intron/exon boundaries were sequenced in both directions. Oligonucleotides were spaced approximately 80-100 bp apart spanning the cDNA. All DNA sequence data was analyzed using the IntelliGenetics program. Southe n and Northern blotting. Genomic DNA was isolated from cell lines as previously described (Amann et al, Genom, 1996., 32:260-265).
  • Northern blots total human tissue, human fetal tissue, and human lymphoid tissue
  • Clontech Piero Alto, CA
  • Hybridizations and washes were performed exactly as described for Southern blotting.
  • These Northern blots were visualized by exposure to Kodak XAR-5 film at -80°C for four days. Equivalent levels of mRNA were confirmed by hybridization to a ⁇ -actin cDNA (data not shown).
  • Fluorescent in situ hybridization (FISH) analysis Bromodeoxyuridine- synchronized and phytohemagglutinin-stimulated peripheral blood lymphocytes from a normal male donor were used as the source of metaphase chromosomes for the chromosomal localization studies.
  • Purified DNA from BAC clones containing the human CASP8 gene (CASP 43 and CASP 44) were labeled for FISH analysis by nick-translation with digoxigenin-11-UTP (Boehringer Mannheim). Slides were baked at 55°C for one hour, RNase treated, denatured in 70% formamide/2X SSC for two minutes at 70°C, and denatured with 100% ethanol.
  • the labeled probe was denatured and hybridized to the chromosomes overnight in the presence of 50% formamide, 10% dextran sulfate, and human COT-1 genomic DNA. Washes were performed at 46°C.
  • one set of metaphase chromosomes was simultaneously hybridized with a biotin labeled genomic heterochromatin specific clone on human chromosome 2 (Oncor, Inc.) Specific hybridization signals were detected by incubating the hybridized slides in fluorescein-conjugated sheep antibodies to digoxigenin (Boehringer Mannheim).
  • CASP8 contains 10 exons spanning ⁇ 30 kb as shown in Figure 1 (GenBank accession numbers for CASP8 sequences are: AF102139J 02146). The 5' UTR is contained in three exons, while the 3' UTR is encoded by one exon. The open reading frame (ORF) begins in exon 3 ( FigurelA). Sequences of exons 3, 4, 5, 6, 7, 8, 9, and 10, including partial sequences of the flanking introns, were determined.
  • Exons 9-10 contain the large and small subunits that constitute the cysteine protease catalytic domain. Much of this cysteine protease domain, including the catalytic cysteine residue (QACXG) (SEQ ED NOJ 1) of the large subunit and the substrate specificity determinants of the small subunit (RNPAEGTW) (SEQ ED NO: 12), are found in exon 9.
  • QACXG catalytic cysteine residue
  • RPAEGTW substrate specificity determinants of the small subunit
  • the structure of the human CASP8 gene is identical to the mouse with regard to the exon/intron organization and exon size (Sakamaki et al, Eur. j. Biochem., 1998, 253:399-405).
  • the sequence of the exon/intron boundaries is shown in Table 1.
  • the structure of the mouse gene, co ⁇ esponding to the ORF portion of the mRNA, is encoded by eight exons. This is identical to the eight exons (i.e., exons 3-10) that encode the human ORF.
  • the 5' UTR of the mouse gene was not analyzed (Sakamaki et al, 1998, supra).
  • BAC 43 and BAC 44 also contain the CASPIO gene, as confirmed by both PCR and limited DNA sequence analysis of the first and last exons of the gene ( Figure 1).
  • the CASPIO gene is located -20-30 kb 5' of the CASP8 gene in the same transcriptional orientation. Southern and Northern blot analysis ofCASP8, and two RFLPs associated with the gene.
  • RFLPs can now be used to track specific alleles associated with CASP8 in families and/or individuals with different genetic diseases/cancer to determine whether this gene is a candidate for mutation.
  • Northern blot analysis of human adult tissues demonstrated that caspase-8 mRNA is expressed fairly ubiquitously (data not shown). Testis, skeletal muscle and kidney express very little caspase-8, while brain appears to express extremely high levels of this mRNA. This is in agreement with previously reported results for the tissue distribution of caspase-8 mRNA (Boldin et al, 1996, supra).
  • lymphoid tissues including spleen, lymph node, thymus, peripheral blood leukocytes (PBLs), bone ma ⁇ ow, and fetal liver, particularly those that undergo significant apoptosis.
  • Northern blot analysis of these tissues shows that caspase-8 is expressed (data not shown). The highest level of expression was found in PBLs, consistent with caspase-8's role in the death of peripheral T cells. Lymph node and spleen also expressed significant levels of caspase-8 mRNA, while thymus, bone marrow, and fetal liver expressed very low levels.
  • CASP8 mRNA expression was also evaluated in fetal tissues (embryonic RNAs, in addition to liver). While human fetal brain expressed undetectable levels of caspase-8 mRNA, and human fetal liver and lung expressed low levels of caspase-8 mRNA, human fetal kidney expressed the highest level of caspase- 8 mRNA (data not shown), similar to what has been reported during the mouse development (Varfolomeev et al, Immunity, 1998, 9:267-276). Chromosome localization of the human CASP8 gene. By fluorescent in situ hybridization (FISH) analysis, the CASP8 gene is localized to human chromosome band 2q33-34 ( Figures 3A and 3B).
  • FISH fluorescent in situ hybridization
  • Neuroblastoma cell lines NB1-8, NB10, and NB 12-21 from St. Jude Children's Research Hospital were tested.
  • Memorial Sloan Kettering neuroblastoma cell lines SK-N-AS, SK-N-F1, and SK-N-DZ, and NIH small-cell lung carcinoma cell lines NCIH 889, NCIH 810, NCIH H69, HCIH H82, and NCIH N417 were analyzed.
  • HeLa and Jurkat lines were used as controls.
  • RNA from each of the indicated neuroblastoma cell lines was examined for the expression of various cell death components: FasL, FasR, FADD, DR-3, FAP, FAF, TRAIL/DR-2, TNFRp55, TRADD, RIP, L32, and GAPDH ( Figure 5).
  • FasL FasR
  • FADD RNA from each of the indicated neuroblastoma cell lines
  • DR-3 RNA from each of the indicated neuroblastoma cell lines
  • FAP FAF
  • TRAIL/DR-2 TNFRp55
  • TRADD RIP
  • L32 L32
  • GAPDH GAPDH
  • caspase-8 is also indicated as a therapeutic tumor suppressor gene.
  • Vectors for expression of caspase-8 will be useful in tumor cells in which caspase-8 has been inactivated.
  • overexpression of caspase-8 in other tumor cells is expected to induce apoptosis and cell death.
  • DNA in 100 microliters was desulphonated by addition of 1/10 volume of freshly prepared 3M NaOH and incubation at 37°C for 15 min, before neutralizing with 166 microliters of ammonium acetate (pH 7.0) and precipitating with 2.5 volumes of ethanol.
  • Bisulfite modified DNA was resuspended in 40 microliters of water.
  • Amplification of the 5' untranslated region of Casp8 gene was performed in 50 microliters reaction mixtures containing 5 microliters of bisulfite treated DNA, dNTPs ,each at 1.25 mM, 300 ng of each primer, IxPCR buffer (Promega, Madison, WE) plus 6.7 mM MgCl 2 and 2.5 units of AmpliTaq polymerase (Promega).
  • Primer sets were designed to produce a 320 bp fragment in the upstream region of Casp8 gene extending from +221 to +541. Wild type primers were used to amplify the co ⁇ esponding region from 100 ng of untreated genomic DNA. Controls without DNA were performed for each set of PCR primers. DNA not treated with bisulfite (unmodified) failed to amplify with either set of methylated or unmethylated specific primers, but readily amplified with the wild type primers- primers specific for the sequence before modification. Primers for methylated -specific bisulfite treated DNA were:
  • Reactions were hot-started at 95 °C for 5 min before the addition of AmpliTaq polymerase.
  • Amplification was done under the following conditions: 35 cycles of 95 °C for 30 sec, 58 c for the unmethylated primers and for the wild-type primers, or 64 °C for the methylated primers, for 30 sec, and 72 °C for 45 sec for all primers. These cycles were followed by 1 cycle of extension at 72 °C for 10 min.
  • Amplification products (10 microliters) were loaded on a 2% agarose gel in TBE buffer, stained with ethidium bromide, and directly visulized under UV illumination.
  • the other primer set retained guanines at positions corresponding to cytosines in CpG dinucleotide sequences specific for methylated genomic DNA (SEQ ED NOS: 29, 30), since these residues are protected from bisulfite modification by methylation ( Figure 6B).
  • primers 31 and 32 for a PCR with bisulfite-treated genomic DNA from the NB cell lines as template the 322-bp CASP8 product was observed in NB3, NB5, NB 15, NB16, and NB21, which express caspase-8, as well as the Jurkat and HeLa positive controls (Figure 6B).
  • DNA sequence analysis of this product from NB16 revealed that all of the sense-strand cytosines within the 322-bp region were replaced with thymidine.
  • a PCR product corresponding predominantly to the unmethylated allele was detected in one of five patient samples, while the remaining two patient samples contained PCR products in reactions amplified with both primer sets.
  • the results listed in Table 3 for all of the remaining NB patient samples were scored in an identical manner.
  • Semi-quantitative PCR was performed to determine whether these patient tumor cells expressed caspase-8 mRNA. For example, S J- 890191, as well as other patient samples with predominantly methylated CASP8 alleles, did not express caspase-8 mRNA, whereas SJ-910008, and other patient samples with predominantly unmethylated, or a mixture of unmethylated and methylated, CASP8 alleles, did express caspase-8 mRNA ( Figures 7B and 7C).
  • CASP8 complete methylation, and therefore silencing, of CASP8 did not occur in six of six late stage (i.e., stage 4) neuroblastoma patient samples that did not contain amplified MYCN genes (Table 3; SJ- 910007, SJ- 970009, POG-687, POG-770, POG-1315, POG-3169, POG-5044). Furthermore, complete methylation of CASP8 occu ⁇ ed in one patient sample (i.e., POG-5217; Table 3) from a stage two neuroblastoma with MYCN amplification.
  • the cell microinjection experiments employed a Fas receptor expression construct (Cheng et al, Science, 1994, 263:1759-1761), a DR3 expression construct (Kitson et al, Nature, 1996, 384:372-375), and an FADD and caspase-8(DN) expression constructs (Persons et al, Blood, 1999, 93:488-499; Yeh et al, Science, 1998, 279:1954-1958).
  • Full-length wild type and dominant negative (DN) caspase-8 cDNAs were subcloned directly into pSP72 (Promega).
  • Wild-type and DN caspase-8 cDNA inserts were then removed by Cla land Sal I digestion from ⁇ SP72 and ligated into the Nsp V and Xho /sites of pMSCVJGFP(M), respectively. Retrovirus supernatants were prepared as previously described (Persons et al, Blood, 1999, 93:488-499). To prevent the spontaneous activation of caspase-8 during transfection, 50 mM zVAD-fink was added into the cell culture medium. 2xl0 6 NB7, NB8 and NB10 cells were prepared the day before transduction in 10-mm culture dishes.
  • Apoptotic assays Cells were collected for annexin V-FITC staining and FACS analysis after the culture media containing zVAD-fink was removed and replaced with normal culture medium for 6 hours in NB8 and NB 10.
  • anti- FAS antibody (lOOng/ml of CH-11) or TNFa (100 ng/ml) plus CHX (1 mg/ml) were then added to the cells and annexin-V-PE (PharMingen) staining performed as previously described (Tang et al, J.Biol.Chem., 1999, 274:7245-7252) .
  • Cell survival assays were performed by standard trypan blue exclusion assay and analysis of nuclear DNA condensation by DAPI staining (Janicke et al, Mol.Cell.Biol., 1994, 14:5661-
  • Viable cells were determined by light and immunofluorescent microscopy after removal of the zVAD-fmk containing culture medium and incubation at 37°C for 0.5, 1, 2, 4 and 6 hrs for the pMSCV-Casp 8(wt) and DN transduced NB8 and NB10 cells.
  • caspase-8 As a target for anti-cancer therapy, it is essential to determine whether the loss of caspase-8 expression in the neuroblastoma cell lines is functionally relevant. All studied NB cell lines express either the Fas or DR3 receptors ( Figure 5, Table 2, and Grenet et al. , Genomics, 1998, 49:385-393), both of which signal through FADD-dependent recruitment of caspase-8 (Kitson et al. , Nature, 1996, 384:372-375; Muzio et al , Cell, 1996, 85:817-827).
  • Fas positive (NB8, NB10) and Fas negative (NB7) cell lines were transduced with a retroviral vector containing either wild-type caspase-8 (pMSCV- Casp8(wt)) or an inactive form of caspase-8 (pMSCV-Casp8(DN); Muzio et al, Cell, 1996, 85:817-827).
  • Caspase-8 expression programmed by the vector was verified by immunoblot analysis ( Figure 8A). Since NB7, the cell line with homozygous CASP8 deletion, does not express Fas receptor, an alternative death receptor was chosen to induce apoptosis.
  • TNFR p55 a Fas-related death receptor
  • CHX TNF- ⁇ and cycloheximide
  • NB8 and NB10 cells resulted in 60-65% cell death in the absence of apoptotic stimuli (Figure 8D).
  • Figure 8E When stable NB8 and NB10 cell populations expressing lower, non- lethal, levels of caspase-8 were selected, they did not undergo significant apoptosis in the absence of an appropriate stimulus ( Figure 8E).
  • Figure 8E addition of agonistic Fas mAb to NB8 culture media resulted in greater than 98% cell death within six hours (Figure 8E). If the potent caspase inhibitor zVAD-fmk (Milligan et al.
  • caspase-8 Although the participation of caspase-8 in tumor cell death induced by chemotherapeutic drug treatment has been considered a controversial issue, it has been suggested that both Fas/CD95-dependent and independent activation of caspase-8 is involved (Landowski et al, Blood, 1999, 94:265-274; Fulda et al, Cancer Res., 1997, 57:3823-3829). In further support of this, we found that NB cell lines (e.g., NB16 and NB21) expressing caspase-8 are more sensitized to doxorubicin-induced apoptosis, whereas cell lines that do not express caspase-8 are not sensitized, regardless of their Fas/CD95 expression status (Table 4).
  • NB cell lines e.g., NB16 and NB21
  • NB7 cells and NB7 cells transduced with pMSCV-Casp ⁇ (wt) were treated with doxorubicin.
  • Doxorubicin-induced neuroblastoma apoptosis has previously been linked to the Fas receptor pathway (Fulda et al, Cancer Res., 1997, 57:3823-3829).
  • the NB7 cell line was chosen since it lacks a functional CASP8 gene ( Figures 4 and 5, Table 2).
  • NB7 cells expressing functional caspase-8 are resensitized to treatment with doxorubicin, demonstrating that caspase-8 expression and activation are capable of accelerating NB7 cell apoptosis and that caspase-8 is functionally relevant to this form of cell death.
  • caspase-8 has been shown to play a pivotal role in apoptosis induced by activated death receptors (Askew et al, Oncogene, 1991, 6: 1915-1922; Kitson et al, Nature, 1996, 384: 372-375), chemotherapeutic drugs (e.g., doxorubicin) and i ⁇ adiation (Friesen et al, Nature Med., 1996, 2: 574-577; Fulda et al, Cancer Res., 1997, 57: 3823-3829). In at least one NB cell line, loss of caspase-8 expression appears to contribute to reduced levels of doxorubicin-induced apoptosis, as well as the dysregulated proliferation, that characterize MYCN amplified neuroblasto

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Abstract

L'invention concerne l'identification d'une activité de suppression de tumeur de la protéine caspase-8 (CASP8), le diagnostic associé, ainsi que des compositions et méthodes thérapeutiques. La découverte de cette activité de suppression de tumeur permet l'obtention de cibles de criblage, de même que, notamment, le criblage de composés déjouant l'inactivation de gènes résultant de la méthylation génomique du promoteur, et elle a permis de constater notamment que CASP8 est inactivée sur le plan fonctionnel dans plus de 90 % de toutes les lignées cellulaires analysées du neuroblastome amplifié MYCN. On a observé l'inactivation de CASP8 par délétion homozygote, délétion hétérozygote, couplées à la mise sous silence de gènes par méthylation, et à la mise sous silence de gènes homozygotes par méthylation. L'invention concerne également une analyse de méthylation par PCR, destinée à mettre en évidence l'inactivation de CASP8.
PCT/US1999/031280 1998-12-31 1999-12-30 Proteine de suppression de tumeur, impliquee dans la transmission des signaux de mort, et methodes diagnostiques, therapeutiques et de criblage basees sur cette proteine WO2000039347A1 (fr)

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WO2002070742A1 (fr) * 2001-03-01 2002-09-12 Epigenomics Ag Procede de mise au point de groupes d'echantillons de genes a des fins de diagnostic et de therapie qui sont bases sur l'expression et l'etat de methylation des genes
EP1283052A1 (fr) * 2001-08-10 2003-02-12 Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts Composition pharmaceutique contenant les caspase-8 et/ou caspase-9 utiles pour surmonter la résistance aux tumeurs induites par la glucocorticoide- et chimiothérapie
CN108431226A (zh) * 2016-01-15 2018-08-21 阿斯利康(瑞典)有限公司 基因修饰测定
CN111378010A (zh) * 2018-12-27 2020-07-07 中国医学科学院药物研究所 靶向caspase-8探针的合成及其在抑制剂筛选方面的应用

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002070742A1 (fr) * 2001-03-01 2002-09-12 Epigenomics Ag Procede de mise au point de groupes d'echantillons de genes a des fins de diagnostic et de therapie qui sont bases sur l'expression et l'etat de methylation des genes
EP1283052A1 (fr) * 2001-08-10 2003-02-12 Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts Composition pharmaceutique contenant les caspase-8 et/ou caspase-9 utiles pour surmonter la résistance aux tumeurs induites par la glucocorticoide- et chimiothérapie
WO2003013591A3 (fr) * 2001-08-10 2003-04-17 Deutsches Krebsforsch Preparations pharmaceutiques contenant de la caspase-8 et/ou de la caspase 9 et permettant de surmonter la resistance des tumeurs a l'apoptose induite par les glucocorticoides et les therapies anticancereuses
CN108431226A (zh) * 2016-01-15 2018-08-21 阿斯利康(瑞典)有限公司 基因修饰测定
CN111378010A (zh) * 2018-12-27 2020-07-07 中国医学科学院药物研究所 靶向caspase-8探针的合成及其在抑制剂筛选方面的应用

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